The goal of this Program Project is the development of new antimalarials effective against drug-resistant strains of Plasmodium. Several existing drugs target the dihydrofolate reductase activity encoded by the parasites as a single polypeptide fused with thymidylate synthase (TS). While this second enzyme has so far not been exploited as a drug target for infectious diseases, the recognized importance of TS as a target for anti-cancer treatment provides us with two decades of medicinal chemistry and pharmacology and three attractive leads to develop potent and species selective TS inhibitors: 1843U89, AG331, and AG337. All three sets of zompounds inhibit the activity of the P. falciparum enzyme in the nM range and 1843U89 has sub-mu M activity against malaria in cells. The goal of this project is to use structure-based methods to gain insight into the interactions between these anti-folates and TS to inform the synthesis of new derivatives with improved potency. The prior state of the art on human and prokaryotic TS demonstrates that rational approaches are very effective. The recent structure of the P. falciparum TS enzyme provides us with the exciting opporttmity to apply these methods to malaria. Specifically, the lab proposes to use computational automated docking and pharmacophore search Iools and structural (NMR) methods of rational structure-based drug design in order to: 1) Identify regions of the malaria enzyme that differ structurally from the human enzyme; 2) Develop a pharmacophore model to be used to screen compound libraries; 3) Use automated docking methods to identify new and chemically Iractable functional groups to append onto the 1843U89, AG331, and AG337 scaffolds to improve their potency and selectivity; 4) Prepare human and Plasmodium TS enzymes in large a mounts and isotopically labeled form to execute NMR studies of ligand binding; 5) Use ligand-directed NMR methods to screen experimentally small molecular weight ligands directed at specific sites within malaria TS; 6) Use protein NMR methods to investigate the structure of the human and Plasmodium TS-ligand complexes and to investigate conformational changes that occur in the active site of the enzyme upon ligand binding. Accomplishment of these goals will facilitate transition of TS-based antifolate drug development from the discovery phase to efficacy in whole organisms.